RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

cyclosporin A (CsA) (10 μM) (d) or BAY11-7082 (5 μM) (e).f Schematic depiction of the putative NFATc1 and NF-κB p65 binding sites in the promoter region of the Rufy4 gene.The region upstream of the Rufy4 transcription initiation sites was divided into four parts, each of which was subcloned into pGL3-Basic luciferase vector (Luc-EV) to generate the Luc-P1 to Luc-P4 Rufy4 promoter constructs.g, h To conduct luciferase-reporter assays, HEK293T cells were co-transfected with Luc-EV or the indicated promoter constructs plus the pcDNA-EV (white) or NFATc1 (g) or NF-κB p65 (h) expression vector (black).The data are presented as mean ± SD of at least three independent experiments.One-way ANOVA with

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Tukey's multiple comparison post hoc test (b), two-tailed unpaired Student's t test (c), or two-way ANOVA with Tukey's multiple comparison post hoc test (d-e, g-h) was used for statistical analysis.**P < 0.01; ***P < 0.001; ****P < 0.0001.Supplementary Figure 2. RUFY4 overexpression in BMMs has no effect on osteoclast formation but increases their bone resorption.Wild-type BMMs that were transduced with EV or RUFY4 were cultured with M-CSF and RANKL on a plastic surface.a The cells were fixed and stained with TRAP.TRAP-positive multinucleated cells (MNCs) were counted (n = 3).Scale bar, 500 μm.b Immunoblot analysis of NFATc1, V-ATPase D2 and active CTSK levels in the cell lysates.The band intensities were determined with ImageJ and normalized to actin (n = 3).Actin served as a loading control.Western blots were probed with the indicated antibodies.c qRT-PCR analysis of Rufy4, Nfatc1, Atp6v0d2 and Ctsk mRNA.Assays were performed in duplicate.d Wild-type BMMs that were transduced with EV or RUFY4 were cultured with M-CSF and RANKL on bone slices.Hematoxylin staining was conducted to visualize the resorption pit area (n = 4).Scale bar, 250 μm.The data are presented as mean ± SD.Two-tailed unpaired Student's t test (a, d) or two-way ANOVA with Tukey's multiple comparison post hoc test (b, c) was used for statistical analysis.*P < 0.05; **P < 0.01; ****P < 0.0001; n.s., not significant.Supplementary Figure 3. Generation and characterization of Rufy4 global knockout mice.a Schematic depiction of the CRISPR/Cas9-mediated Rufy4 gene deletion.b PCR analysis of Rufy4 +/+ , Rufy4 +/-, and Rufy4 -/-mice.c qRT-PCR analysis of the Rufy4 mRNA in Rufy4 +/+ and Rufy4 -/-osteoclasts generated from BMMs with M-CSF and RANKL.d Bodyweight changes in Rufy4 +/+ and Rufy4 -/-mice (n = 7 mice per group).e qRT-PCR analysis of Rufy1, Rufy2, Rufy3 and Fyco1 mRNA in Rufy4 +/+ and Rufy4 -/-osteoclasts generated from BMMs with M-CSF and RANKL.The data are presented as mean ± SD.Two-way ANOVA with Tukey's multiple comparison post hoc test (c, e) or two-tailed unpaired Student's t test (d) was used for statistical analysis.****P < 0.0001; n.s., not significant.or autophagy signaling.a, b Gating strategy for detection of BMDCs after Rufy4 +/+ and Rufy4 -/-bone marrow cells were treated without (a) or with (b) LPS.Representative flow cytometry plots of mature BMDCs (CD11c + CD11b + MHCII + ) are shown and the mature BMDC frequencies are plotted (n = 3).c qRT-PCR analysis of the cd86 mRNA in Rufy4 +/+ and Rufy4 -/-BMDCs generated from bone marrow cells with LPS.d Immunoblot analysis of the expression and activation of the autophagy signaling molecules Atg5, Atg7, Atg12, p62, p-mTOR, mTOR, LC3B, p-Beclin1, and Beclin1 in mature Rufy4 +/+ and Rufy4 -/-osteoclasts that were treated with or without the autophagy inducer, rapamycin (10 nM).The band intensities were determined with ImageJ and normalized to actin (n = 3).Actin served as a loading control.Western blots were probed with the indicated antibodies.The data are presented as mean ± SD.Two-tailed unpaired Student's t test (a, b) or two-way ANOVA with Tukey's multiple comparison post hoc test (c, d) was used for statistical analysis.n.s., not significant.theperinuclear level (upper level expression as described in Fig.2f).Mean intensity per cell was quantified (n = 3).Scale bars, 10 μm.c, d Immunofluorescence staining of early endosome antigen 1 (EEA1) in osteoclasts at the perinuclear (c) or bone surface level (d).Mean intensity per cell or actin ring was quantified (n = 3).Scale bars, 10 μm.e, f Immunofluorescence staining of transferrin receptor (TfR1) in osteoclasts at the perinuclear (e) or bone surface level (f).Mean intensity per cell or actin ring was quantified (n = 3).Scale bars, 10 μm.g MFI of Alexa Fluor 546 dextran in mature Rufy4 +/+ and Rufy4 -/- osteoclasts (n = 6).h qRT-PCR analysis of Tfeb, Scarb, Lamp2 and Cd63 mRNA in Rufy4 +/+ and Rufy4 -/-osteoclasts.The data are presented as mean ± SD of at least three independent experiments.Two-tailed unpaired Student's t test (a-g) or two-way ANOVA with Tukey's multiple comparison post hoc test (h) was used for statistical analysis.n.s., not significant.Immunofluorescence staining of Rab7 (a) or LAMP2 (b) in HeLa cells that were transfected with RUFY4-EGFP.Scale bar, 10 μm.c Schematic depiction of the RUFY4 deletion mutants.d, e Immunofluorescence staining of Rab7 (d) or LAMP2 (e) in HeLa cells that were transfected with EV-EGFP, RUFY4-EGFP, or the ΔRUN-EGFP, ΔFYVE-EGFP, or ΔCC-EGFP mutants.Pearson's correlation coefficient is a statistic for quantifying the colocalization of each construct with Rab7 or LAMP2 (n = 3).Scale bars, 10 μm.f, g HEK293T cells were transfected with the ΔCC mutant of RUFY4 and subjected to co-IP analyses of its association with Rab7 (f) or LAMP2 (g) (n = 3).Western blots were probed with the indicated antibodies.h BMMs from Rufy4 -/-mice were transduced with EV, RUFY4, or the ΔRUN, ΔFYVE, or ΔCC mutants, treated with M-CSF and RANKL on a plastic surface.The cells were then stained with TRAP and TRAP-positive MNCs were counted (n = 3).Scale bar, 500 μm.The data are presented as mean ± SD.One-way ANOVA with Tukey's multiple comparison post hoc test was used for statistical analysis.****P < 0.0001; n.s., not significant.

Table S2 .
List of antibodies for immunofluorescence staining

Table S3 .
List of antibodies for flow cytometry

Table S4 .
List of primers for cloning (Enzyme sites are in bold.)

Table S5 .
List of primers for qRT-PCR